There are approximately 20 million new sexually transmitted infections (STIs) in the U.S. each year that add $16 billion in medical costs to the healthcare system. One approach to prevention of STIs in women that has not been fully explored is harnessing the protective features of the vaginal microbiome. The vaginal microbiota play an important role in preventing colonization by pathogenic organisms. Vaginal Lactobacillus spp. provide broad-spectrum antimicrobial activity in part through their production of lactic acid, which creates an acidic and hostile environment to pathogens. To date, only studies based on bacterial cultivation techniques or microscopic evaluation are available to describe the temporal association between the vaginal bacteria and risk of STI. However, a majority of microbial species (>90%) resist cultivation and microscopy only provides morphological information. Complete characterization of the vaginal microbiota requires molecular approaches as we propose. We have shown using metagenomic analysis that specific genotypes of Lactobacillus spp. are associated with both chlamydial infection and vaginal microbiota instability. We are putting forth a novel hypothesis that, in addition to a low-Lactobacillus state, specific Lactobacillus genotypes are associated with increased risk for STIs, and that the mixture of D- and L- lactate isomers produced by Lactobacillus spp. differentially affects STI risk. Inflammation may cause destruction of the vaginal epithelium, allowing pathogens to access deeper tissues, and therefore, we also hypothesize that local immune responses associated with specific vaginal microbiota may facilitate STIs. We seek to utilize archived cervicovaginal lavage (CVL) samples collected from the 1999 NIH Longitudinal Study of Vaginal Flora in which 3,620 women were followed for one year with quarterly assessments. A total of 681 women were observed to acquire an incident Chlamydia trachomatis (CT), Neisseria gonorrhea (GC), or Trichomonas vaginalis (TV) genital infection. The repository samples provide a unique opportunity to characterize the vaginal microbiome prior to acquisition of STIs in a well-powered study. We will conduct a nested case-control study with incidence density sampling. Cases will be defined as women who had an incident STI and we will analyze the sample at the visit prior to the first STI detection. Controls will be matched on age, ethnicity and visit number to cases and will be women who did not develop a STI by the time of the case visit. Specific aims are to (1) Evaluate the association between vaginal microbiota and incidence of CT, GC, and TV genital infection using 16S rRNA gene analysis; (2) Compare levels of D- and L-lactic acid isomers in cases and controls; (3) Investigate if specific strains of Lactobacillus (or other bacterium) are associated with STI risk using metagenomic analysis; (4) Determine if cervicovaginal immune responses independently predict incident STIs. All four aims will be modeled in the context of comprehensive behavioral data. This study will provide a functional understanding of the vaginal microbiome's role in STI protection and may reveal novel targets to prevent STIs and improve women's health.